Large-scale consumption of the traditional, fossil fuels (petroleum-based fuels) in the last few decades has contributed to high levels of pollution. Moreover, the realisation that the world stock of petroleum is not boundless, combined with the growing environmental awareness, has stimulated new initiatives to investigate the feasibility of alternative fuels such as ethanol, which could realise a 60-90% decrease in CO2 production. Although biomass-derived ethanol may be produced by fermentation of hexose sugars that are obtained from many different sources, so far, however, the substrates for industrial scale production or fuel alcohol are cane sugar and corn starch. The drawback of these substrates are the high costs.
Expanding fuel ethanol production requires the ability to use lower-cost feedstocks. Presently, only lignocellulosic feedstock from plant biomass would be available in sufficient quantities to substitute the crops used for ethanol production. The major fermentable sugars from lignocellulosic materials are glucose and xylose, constituting respectively about 40% and 25% of lignocellulose. However, most yeasts that are capable of alcoholic fermentation, like Saccharomyces cerevisiae, are not capable of using xylose as a carbon source. Additionally, no organisms are known that can ferment xylose to ethanol with both a high ethanol yield and a high ethanol productivity. To enable the commercial production of ethanol from lignocellulose hydrolysate, an organism possessing both these properties would be required. Thus it is an object of the present invention to provide for a yeast that is capable of both alcoholic fermentation and of using xylose as a carbon source.
D-xylose is metabolisable by numerous microorganisms such as enteric bacteria, some yeasts and fungi. In most xylose-utilising bacteria, xylose is directly isomerised to D-xylulose by xylose (glucose) isomerase (XI). Filamentous fungi and yeasts, are however not capable of this one-step isomerisation and first reduce xylose to xylitol by the action of xylose reductase (XR) after which the xylitol is converted to xylulose by xylitol dehydrogenase (XDH). The first step requires NAD(P)H as a co-factor whereas the second step requires NAD+. The xylulose that is produced subsequently enters the pentose phosphate pathway (PPP) after it is phosphorylated by xylulose kinase (XK). Anaerobic fermentation of xylose to ethanol is not possible in organisms with a strictly NADPH dependent xylose reductase (XR). This is because xylitol dehydrogenase (XDH) is strictly NAD+ dependent resulting in a redox imbalance (i.e., NAD+ depletion). To solve the redox imbalance under anaerobic conditions, the organism produces by-products such as glycerol and xylitol. Similarly, aerobic production ofβ-lactams on xylose is also negatively influenced as compared to β-lactam production on glucose. A likely cause for these low yields again are a relatively high demand of reducing equivalents in the form of NADPH in this route, compared to the use of glucose (W. M. van Gulik et al. Biotechnol. Bioeng. Vol. 68, No. 6, Jun. 20, 2000).
Over the years many attempts have been made to introduce xylose metabolism in S. cerevisiae and similar yeasts, as reviewed in Zaldivar et al. (2001, Appl. Microbiol. Biotechnol. 56: 17-34). One approach concerns the expression of at least genes encoding a xylose (aldose) reductase and a xylitol dehydrogenase, e.g. the XYL1 and XYL2 of Pichia stipitis, in S. cerevisiae (U.S. Pat. No. 5,866,382; WO 95/13362; and WO 97/42307). Although this approach enables growth of S. cerevisiae on xylose, it generally suffers from a low ethanol productivity and/or yield as well as a high xylitol production, mainly as a result of the redox imbalance between XR and XDH.
The expression of a XI in S. cerevisiae or related yeast or in filamentous fungi would circumvent the redox imbalance and consequent xylitol production and excretion. Xylose isomerase genes from several bacteria have been inserted in S. cerevisiae, however, expression of mesophilic prokaryotic XIs in S. cerevisiae did not lead to active XI (Amore and Hollenberg, 1989, Nucleic Acids Res. 17: 7515; Amore et al., 1989, Appl. Microbiol. Biotechnol. 30: 351-357; Chan et al., 1986, Biotechnol. Lett 8: 231-234; Chan et al., 1989, Appl. Microbiol. Biotechnol. 31: 524-528; Ho et al., 1983, Fed. Proc. Fed. Am. Soc. Exp. Biol. 42: 2167; Hollenberg, 1987, EBC-Symposium on Brewer's Yeast, Helsinki (Finland), Nov. 24-25, 1986; Sarthy et al., 1987, Appl. Environ. Microbiol. 53: 1996-2000; Ueng et al., 1985, Biotechnol. Lett. 7: 153-158). Nevertheless, two XIs from thermophilic bacteria expressed in S. cerevisiae showed a specific activity of 1 μmol per minute per mg−1 at 85° C. (Bao et al., 1999, Weishengwu-Xuebao 39: 49-54; Walfridson et al., 1996, Appl. Environ. Microbiol. 61: 4184-4190). However, at physiological temperature for S. cerevisiae (20-35° C.) only a few percent of this activity is left, which is not sufficient for efficient alcoholic fermentation from xylose. Thus, there is still a need for nucleic acids encoding an XI that can be expressed in yeasts to provide sufficient XI activity under physiological conditions to allow for the use of xylose as carbon source.